Therapeutic preparation for inhalation

ABSTRACT

A therapeutic preparation for inhalation which comprises insulin and a substance which enhances the absorption of insulin in the lower respiratory tract, is provided in the form of a powder preparation suitable for inhalation.

This invention relates to a therapeutic preparation of insulin, which issuitable for inhalation.

BACKGROUND OF THE INVENTION

Insulin plays a central role in the regulation of carbohydrate, fat, andprotein metabolism in the body. Diabetes mellitus (commonly referred tosimply as diabetes) is a disease characterized by disregulation ofmetabolism, particularly glucose metabolism. In normal individuals, arise in blood glucose levels (such as that which occurs immediatelyfollowing eating) triggers the islet beta cells of the pancreas tosecrete insulin, a peptide hormone, into the bloodstream. The insulinbinds to insulin receptors located on a number of cell types, notablymuscle cells, and thereby signals the cells to increase the rate ofglucose uptake into the cells. As the blood glucose returns to normalpre-prandial levels, the amount of insulin in the blood also drops. Inthe absence of insulin, blood glucose levels would rise to dangerouslyhigh levels (a condition termed hyperglycemia), possibly resulting indeath. Too much insulin causes abnormally low blood glucose levels(hypoglycemia), which is also dangerous and possibly fatal. In a normalindividual, built-in feedback loops regulating the secretion of insulinand its clearance from the systemic circulation prevent bothhyperglycemic and hypoglycemic conditions from occurring.

Diabetes mellitus is a disease affecting about 3% of the population ofSweden. Of these 3%, approximately 20% suffer from Type I diabetes, andthe remainder from Type II diabetes.

Type I diabetes, or insulin-dependent diabetes mellitus (IDDM), usuallybegins in childhood. It is characterized by atrophy of the pancreaticbeta cells, resulting in a decrease or cessation of insulin production,and leaving the patient dependent on exogenous insulin for survival.

The more common Type II diabetes, or non-insulin-dependent diabetesmellitus (NIDDM), generally occurs in patients older than 40 years.These patients may, at least initially, have normal or even high levelsof insulin in their blood, but exhibit an abnormally low rate ofcellular uptake of glucose in response to insulin. Although Type IIdiabetes often can be treated by controlling the patient's diet,administration of exogenous insulin to supplement that secreted by thepatient's beta cells may also prove necessary.

Insulin cannot be orally administered in effective doses, since it israpidly degraded by enzymes in the gastrointestinal tract and low pH inthe stomach before it can reach the bloodstream. The standard method ofadministration is by subcutaneous injection of an isotonic solution ofinsulin, usually by the patient him/herself. The necessity for injectioncauses a great deal of inconvenience and discomfort to many sufferers,and local reactions can occur at the injection site. In addition thereis an abnormal, non-physiological, plasma concentration profile forinjected insulin. This abnormal plasma concentration profile isundesirable and increases the risk of side effects related to the longterm treatment of diabetes.

Because of these disadvantages, there is a need for insulin in a formwhich is administrable other than by injection. In attempts to producesuch different forms of insulin, various proposals have been made. Forexample, products for nasal, rectal and buccal administration have beensuggested, with much effort being concentrated on products for nasaladministration. Nasal administration is however problematic and permitsonly a very low bioavailability. Pulmonary delivery of systemicallyactive drugs has gained increasing interest over the last years, andsome investigations have included the pulmonary delivery of insulin.Most of these are concerned with solutions or suspensions for pulmonarydelivery for example by nebulisers and pressurised metered doseinhalers, and all have met with limited success.

THE INVENTION

We have now found that insulin can be included in a dry powderpreparation for inhalation also including a substance which enhances theabsorption of insulin in the lung, from which preparation the insulinmay be absorbed in a therapeutically acceptable rate and amount. By"enhances absorption" is meant that the amount of insulin absorbed intothe systemic circulation in the presence of the enhancer is higher thanthe amount absorbed in the absence of enhancer.

According to this invention therefore, there is provided a therapeuticpreparation comprising active compounds (A) insulin, and (B) a substancewhich enhances the absorption of insulin in the lower respiratory tract,which preparation is in the form of a dry powder suitable for inhalationin which at least 50% of the total mass of active compounds consists of(a) primary particles having a diameter of less than about 10 microns,for example between 0.01 and 10 microns and preferably between 1 and 6microns, or (b) agglomerates of said particles.

The therapeutic preparation of the present invention may contain onlythe said active compounds or it may contain other substances, such as apharmaceutically acceptable carrier. This carrier may largely consist ofparticles having a diameter of less than about 10 microns so that atleast 50% of the resultant powder as a whole consists of optionallyagglomerated primary particles having a diameter of less than about 10microns; alternatively the carrier may largely consist of much biggerparticles ("coarse particles"), so that an "ordered mixture" may beformed between the active compounds and the said carrier. In an orderedmixture, alternatively known as an interactive or adhesive mixture, finedrug particles (in this invention, the active compounds) are fairlyevenly distributed over the surface of coarse excipient particles (inthis invention, the pharmaceutically acceptable carrier). Preferably insuch case the active compounds are not in the form of agglomerates priorto formation of the ordered mixture. The coarse particles may have adiameter of over 20 microns, such as over 60 microns. Above these lowerlimits, the diameter of the coarse particles is not of criticalimportance so various coarse particle sizes may be used, if desiredaccording to the practical requirements of the particular formulation.There is no requirement for the coarse particles in the ordered mixtureto be of the same size, but the coarse particles may advantageously beof similar size within the ordered mixture. Preferably, the coarseparticles have a diameter of 60-800 microns.

In a particular embodiment therefore this invention provides atherapeutic preparation of insulin and a substance which enhances theabsorption of insulin in the lower respiratory tract, which preparationis in the form of a dry powder preparation suitable for inhalation ofwhich at least 50% by mass consists of (a) particles having a diameterof less than about 10 microns or (b) agglomerates of said particles; ina further particular embodiment, the invention provides a therapeuticpreparation comprising insulin, a substance which enhances theabsorption of insulin in the lower respiratory tract, and apharmaceutically acceptable carrier, which preparation is in the form ofa dry powder suitable for inhalation of which at least 50% by massconsists of (a) particles having a diameter of less than about 10microns, or (b) agglomerates of said particles; and in a still furtherparticular embodiment this invention provides a therapeutic preparationcomprising active compounds (A) insulin and (B) a substance whichenhances the absorption of insulin in the lower respiratory tract,wherein at least 50% of the total mass of active compounds (A) and (B)consists of particles having a diameter of less than about 10 microns,and a pharmaceutically acceptable carrier, which preparation is in theform of a dry powder preparation suitable for inhalation in which anordered mixture may be formed between the active compounds and thepharmaceutically acceptable carrier.

Preferably at least 60% such as at least 70% or at least 80% and morepreferably at least 90% of the total mass of active compounds (A) and(B) consists of particles having a diameter of less than about 10microns, or of agglomerates of such particles, and, when the dry powderpreparation comprises carrier other than when an ordered mixture isdesired, preferably at least 60% such as at least 70% or at least 80%and more preferably at least 90% by mass of the total dry powderconsists of particles having a diameter of less than about 10 microns,or of agglomerates of such particles.

While the dry powder for inhalation, whether with or withoutpharmaceutically acceptable carrier, may contain agglomerates ofparticles as indicated above, at the time of inhalation any agglomeratesshould be substantially deagglomerated yielding a powder of which atleast 50% consists of particles having a diameter of up to 10 microns.The agglomerates can be the result of a controlled agglomeration processor they may simply be the result of the intimate contact of the powderparticles. In either case it is essential that the agglomerates arecapable of being de-agglomerated e.g. by mechanical means in the inhaleror otherwise, into the aforesaid particles. Agglomerates are in generalpreferably not formed in the ordered mixture. In the case of an orderedmixture, the active compounds should be released from the largeparticles preferably upon inhalation, either by mechanical means in theinhaler or simply by the action of inhalation, or by other means, theactive compounds then being deposited in the lower respiratory tract andthe carrier particles in the mouth.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph comparing the effect of inhaled insulin plus enhancerto the effect of inhaled insulin without enhancer in blood glucoselevels in a dog.

FIG. 2 is a graph comparing the effect of inhaled insulin plus enhancerto the effect of inhaled insulin without enhancer on blood glucoselevels in a second dog.

FIG. 3 is a graph comparing plasma levels of insulin after inhalation ofthree different formulations of insulin and enhancer in a dog.

FIG. 4 is a graph comparing plasma levels of insulin after inhalation ofthree different formulations of insulin and enhancer in a second dog.

FIG. 5 is a graph comparing blood glucose levels following inhalation ofthree different formulations of insulin and enhancer in a dog.

FIG. 6 is a graph comparing blood glucose levels following inhalation ofthree different formulations of insulin and enhancer in a second dog.

FIG. 7 is a graph showing the effect of three different concentrationsof enhancer on transport of mannitol across a Caco-2 cell monolayer.

FIG. 8 is a graph showing the effect of three different concentrationsof enhancer on transport of mannitol across a Caco-2 cell monolayer, inthe presence of insulin.

FIG. 9 is a graph comparing plasma insulin levels following inhalationof insulin alone, or insulin:sodium caprate 75:25 or 90:10.

DETAILED DESCRIPTION

Any biologically active form or derivative of insulin may be used in thepresent invention. For example bovine, porcine, or biosynthetic orsemisynthetic human insulin, or a biologically active derivative ofhuman insulin ("modified insulin"), for example having certain aminoacid substitutions as taught by Brange et al in "Diabetes Care" 13:923,1990, may be used. Modified insulins are developed in order to improvevarious properties, for example to improve stability or give an improvedpharmokinetic profile (i.e. improved profile of absorption through theepithelial membranes). The insulin should have a low zinc content, sincezinc lowers the solubility of insulin, probably reducing the absorptionrate, and also because zinc may form undesirable insoluble precipitateswith certain of the enhancer substances for use in the presentinvention. In addition the insulin should be in the form of a dry powderwhich dissolves rapidly in aqueous solution.

The substance which enhances the absorption of insulin in the lung,hereinafter referred to as the enhancer, can be any of a number ofcompounds which act to enhance absorption through the layer ofepithelial cells in the lower respiratory tract, and into the adjacentpulmonary vasculature. The enhancer can accomplish this by any ofseveral possible mechanisms:

(1) Enhancement of the paracellular permeability of insulin by inducingstructural changes in the tight junctions between the epithelial cells.

(2) Enhancement of the transcellular permeability of insulin byinteracting with or extracting protein or lipid constituents of themembrane, and thereby perturbing the membrane's integrity.

(3) Interaction between enhancer and insulin which increases thesolubility of insulin in aqueous solution. This may occur by preventingformation of insulin aggregates (dimers, trimers, hexamers), or bysolubilizing insulin molecules in enhancer micelies.

(4) Decreasing the viscosity of, or dissolving, the mucus barrier liningthe alveoli and passages of the lung, thereby exposing the epithelialsurface for direct absorption of the insulin.

Enhancers may function by only a single mechanism set forth above, or bytwo or more. An enhancer which acts by several mechanisms is more likelyto promote efficient absorption of insulin than one which employs onlyone or two. For example, surfactants are a class of enhancers which arebelieved to act by all four mechanisms listed above. Surfactants areamphiphilic molecules having both a lipophilic and a hydrophilic moiety,with varying balance between these two characteristics. If the moleculeis very lipophilic, the low solubility of the substance in water maylimit its usefulness. If the hydrophilic part overwhelmingly dominates,however, the surface active properties of the molecule may be minimal.To be effective, therefore, the surfactant must strike an appropriatebalance between sufficient solubility and sufficient surface activity.

Another surfactant property that may be of importance is the net chargeof the surfactant at the pH value in the lung (approximately 7.4). Theisoelectric pH of insulin is 5.5. At pH 7.4, insulin has a negative netcharge. This results in an electrostatic repulsion between insulinmolecules, which in turn prevents aggregation and thereby increases thesolubility. If the surfactant also is negatively charged, yet caninteract with insulin by, for example, hydrophobic interactions,additional repulsion among the insulin molecules will occur. Therefore,an anionic surfactant will possess the additional advantage (compared tothose having neutral or net positive charge at physiological pH) ofenhancing absorption by helping stabilize insulin in the monomericstate.

A number of different compounds potentially useful as enhancers in themethods of the invention were tested in rats, as described in Example 5below. Other substances with known absorption-enhancing properties, orwith physical characteristics which make them likely candidates for usein the method of the invention, can be readily tested by one of ordinaryskill in that in vivo assay, or alternatively in the in vitro assaydescribed in Example 6.

It is possible that a combination of two or more enhancer substancesalso gives satisfactory results. The use of such a combination in themethod of the invention is considered to be within the invention.

An enhancer useful in the methods of the invention will combineeffective enhancement of insulin absorption with (1) lack of toxicity inthe concentrations used and (2) good powder properties, i.e., lack of asticky or waxy consistency in the solid state. Toxicity of a givensubstance can be tested by standard means, such as by the MTT assay, forexample as described in Int. J. Pharm., 65 (1990), 249-259. The powderproperties of a given substance may be ascertained from published dataon the substance, or empirically.

One very promising type of enhancer is the salt of a fatty acid. It hasbeen found that the sodium salts of saturated fatty acids of carbonchain length 10 (i.e., sodium caprate), 12 (sodium laurate) and 14(sodium myristate) perform well in the method of the invention. Thepotassium and lysine salts of capric acid have also been found to beeffective in the method of the invention. If the carbon chain length isshorter than about 10, the surface activity of the surfactant may be toolow, and if the chain length is longer than about 14, decreasedsolubility of the fatty acid salt in water limits its usefulness.

Most preferably in the present invention the substance which enhancesthe absorption of insulin in the lower respiratory tract is sodiumcaprate.

In a particularly preferred embodiment therefore, this inventionprovides a therapeutic preparation comprising active compounds (A)insulin and (B) sodium caprate, in the form of a dry powder suitable forinhalation in which at least 50% of the total mass of active compounds(A) and (B) consists of (a) primary particles having a diameter of lessthan about 10 microns, for example between 0.01 and 10 microns andpreferably between 1 and 6 microns, or (b) agglomerates of suchparticles; specifically, in this particularly preferred embodiment thisinvention provides:

a therapeutic preparation of insulin and sodium caprate, whichpreparation is in the form of a dry powder suitable for inhalation ofwhich at least 50% by mass consists of (a) particles having a diameterof less than about 10 microns or (b) agglomerates of said particles;

a therapeutic preparation comprising insulin, sodium caprate and apharmaceutically acceptable carrier, which preparation is in the form ofa dry powder suitable for inhalation of which at least 50% by massconsists of (a) particles having a diameter of less than about 10microns, or (b) agglomerates of said particles; and

a therapeutic preparation comprising active compounds (A) insulin and(B) sodium caprate, wherein at least 50% of the total mass of activecompounds (A) and (B) consists of particles having a diameter of lessthan about 10 microns, and a pharmaceutically acceptable carrier, whichpreparation is in the form of a dry powder preparation suitable forinhalation in which an ordered mixture may be formed between the activecompounds and the pharmaceutically acceptable carrier.

Different counterions may change the solubility of the saturated fattyacid salt in water, such that an enhancer having a carbon length otherthan 10-14 would prove even more advantageous than the enhancersspecifically mentioned hereinabove. Salts of unsaturated fatty acids mayalso be useful in the present invention since they are more watersoluble than salts of saturated fatty acids, and can therefore have alonger chain length than the latter and still maintain the solubilitynecessary for a successful enhancer of insulin absorption.

Bile salts and bile salt derivatives were tested for usefulness asenhancers in the method of the present invention. All of those tested(sodium salts of ursodeoxycholate, taurocholate, glycocholate, andtaurodihydrofusidate) effectively enhance insulin absorption in thelung.

Phospholipids were also tested as enhancers. It was found that asingle-chain phospholipid (lysophospatidylcholine) was an effectiveenhancer, while two double-chain phospholipids(dioctanoylphosphatidylcholine and didecanoylphosphatidylcholine) werenot. This may be explained by the fact that the double-chainphospholipids are much less soluble in water than their single-chaincounterparts; however, it is reasonable to expect that double-chainphospholipids of shorter chain length, having greater water-solubilitythan their longer chain counterparts, will be of use as enhancers in thepresent invention so that both single- and double-chain phospholipidsmay be used.

One glycoside, octylglucopyranoside, was tested as an enhancer in thepresent invention and was found to have some absorption enhancingproperties. Other alkyl glycosides such as thioglucopyranosides andmaltopyranosides would also be expected to exhibit absorption enhancingproperties in the methods of the present invention.

The cyclodextrins and derivatives thereof effectively enhance the nasalabsorption of insulin, and may function similarly in the lung.Dimethyl-β-cyclodextrin has been tested in the method of the presentinvention and has been found to have an absorption enhancing effect.

Other potentially useful surfactants are sodium salicylate, sodium5-methoxysalicylate, and the naturally occurring surfactants such assalts of glycyrrhizine acid, saponin glycosides and acyl carnitines.

For ionic enhancers (e.g., the anionic surfactants described above), thenature of the counterion may be important. The particular counterionselected may influence the powder properties, solubility, stability,hygroscopicity, and local/systemic toxicity of the enhancer or of anyformulation containing the enhancer. It may also affect the stabilityand/or solubility of the insulin with which it is combined. In general,it is expected that monovalent metallic cations such as sodium,potassium, lithium, rubidium, and cesium will be useful as counterionsfor anionic enhancers. Ammonia and organic amines form another class ofcations that is expected to be appropriate for use with anionicenhancers having a carboxylic acid moiety. Examples of such organicamines include ethanolamine, diethanolamine, triethanolamine,2-amino-2-methylethylamine, betaines, ethylenediamine,N,N-dibensylethylenetetraamine, arginine, hexamethylenetetraamine,histidine, N-methylpiperidine, lysine, piperazine, spermidine, spermineand tris(hydroxymethyl)aminomethane.

Since effective enhancement of insulin absorption in the lung wasobserved for a number of the enhancers tested, it is expected that manymore will be found which also function in this manner. Starchmicrospheres effectively enhance the bioavailability of insulindelivered via the nasal membranes and were tested as an enhancer in themethods of the invention. Although they proved to be of little use fordelivery via the pulmonary route in the animal model utilized herein, itis thought that this was mainly due to technical difficulties which, ifovercome, may lead to successful delivery via the pulmonary route.Chelators are a class of enhancers that are believed to act by bindingcalcium ions. Since calcium ions help maintain the dimensions of thespace between cells and additionally reduce the solubility of insulin,binding of these ions would in theory both increase the solubility ofinsulin, and increase the paracellular permeability of insulin. Althoughone chelator tested, the sodium salt of ethylenediaminetetraacetic acid(EDTA), was found to be ineffective in enhancing absorption of insulinin the rat model tested, other calcium ion-binding chelating agents mayprove to be more useful.

In general, it is desirable to keep the ratio of insulin to enhancer ashigh as possible, within the range that permits fast and efficientenhancement of insulin absorption. This is important in order tominimize the risk of adverse effects, both local and systemic,attributable to the enhancer. The optimal ratio of insulin to enhancercan be ascertained for any given enhancer by testing various proportionsin in vivo models such as described herein. For example, insulin wascombined with sodium caprate in the following w/w proportions: 50/50,75/25, 82.5/17.5, and 90/10. Significant improvement in absorption ofinsulin was obtained with 50% and 25% sodium caprate; 10% gave poorimprovement in absorption, and the results with 17.5% were intermediate.This indicates that the lowest effective concentration of sodium capratefor use in the methods of the invention is approximately 15-25%, andprobably 20-25%. Other enhancers may have higher or lower optimalconcentrations relative to insulin, and each individual enhancer musttherefore be separately tested. Based upon the above results, however,it is expected that the optimal proportion of a surfactant type ofenhancer will generally be between 10 and 50% of the insulin/enhancermixture, for example between 15% and 50% such as between 25% and 50%. Itshould be noted that the above proportions represent the proportion ofenhancer relative solely to insulin, and do not take into account anycarrier or other additive which may be added, for example to improve thepowder properties of the formulation.

The amount of insulin absorbed according to the present invention can besignificantly higher than the amount absorbed in the absence ofenhancer. In Example 4 herein it is shown that a therapeutic preparationaccording to the present invention, when inhaled, exhibits abioavailability well over three times greater than that of an inhaledpreparation of insulin alone.

Preferably the amount of insulin absorbed according to the presentinvention is significantly (p<0.05) higher than the amount absorbed inthe absence of enhancer.

As stated hereinabove, additive substances commonly included intherapeutic preparations, such as pharmaceutically acceptable carriers,may be included in the theraputic preparation of the present invention.Additive substances may be included for example in order to dilute thepowder to an amount which is suitable for delivery from the particularintended powder inhaler; to facilitate the processing of thepreparation; to improve the powder properties of the preparation; toimprove the stability of the preparation, e.g. by means of antioxidantiaor pH-adjusting compounds; or to add a taste to the preparation. Anyadditive should not adversely affect the stability of the insulin orabsorption enhancer, or disadvantageously interfere with the insulinabsorption. It should also be stable, not hygroscopic, have good powderproperties and have no adverse effects in the airways. As examples ofpotential additives may be mentioned mono-, di-, and polysaccharides,sugar alcohols and other polyols, such as for example lactose, glucose,raffinose, melezitose, lactitol, maltitol, trehalose, sucrose, mannitoland starch. As reducing sugars such as lactose and glucose have atendency to form complexes with proteins, non-reducing sugars such asraffinose, melezitose, lactitol, maltitol, trehalose, sucrose, mannitoland starch may be preferred additives for use in the present invention.Depending upon the inhaler to be used, the total amount of suchadditives may vary over a very wide range. In some circumstances littleor no additive would be required, whereas for example in the case of aninhaler requiring large powder volumes for operation, a very highpercentage of the therapeutic preparation could consist of additive. Theamount of additive desirable would be easily determined by a personskilled in the art according to particular circumstances.

A useful mechanism for delivery of the powder into the lungs of apatient is through a portable inhaler device suitable for dry powderinhalation. Many such devices, typically designed to deliverantiasthmatic or antiinflammatory agents into the respiratory system,are on the market. Preferably the device is a dry powder inhaler of adesign which provides protection of the powder from moisture and has norisk from occasional large doses; in addition as many as possible of thefollowing are desired: protection of the powder from light; highrespirable fraction and high lung deposition in a broad flow rateinterval; low deviation of dose and respirable fraction; low retentionof powder in the mouthpiece; low adsorption to the inhaler surfaces;flexibility in dose size; and low inhalation resistance. The inhaler ispreferably a single dose inhaler although a multi dose inhaler,preferably such as a multi dose, breath actuated, dry powder inhaler formultiple use, may also be employed. Preferably the inhaler used is aunit dose, breath actuated, dry powder inhaler for single use.

The described powder preparation can be manufactured in several ways,using conventional techniques. It may be necessary to micronize theactive compounds and if appropriate (i.e. where an ordered mixture isnot intended) any carrier in a suitable mill, for example in a jet millat some point in the process, in order to produce primary particles in asize range appropriate for maximal deposition in the lower respiratorytract (i.e., under 10 μm). For example, one can dry mix insulin andenhancer powders, and carrier where appropriate, and then micronize thesubstances together; alternatively, the substances can be micronizedseparately, and then mixed. Where the compounds to be mixed havedifferent physical properties such as hardness and brittleness,resistance to micronisation varies and they may require differentpressures to be broken down to suitable particle sizes. When micronisedtogether, therefore, the obtained particle size of one of the componentsmay be unsatisfactory. In such case it would be advantageous tomicronise the different components separately and then mix them.

It is also possible first to dissolve the components including, where anordered mixture is not intended, any carrier in a suitable solvent, e.g.water, to obtain mixing on the molecular level. This procedure alsomakes it possible to adjust the pH-value to a desired level. It is knownthat the nasal absorption of insulin is affected by the pH-value of thepreparation, with increasing absorption when moving either up or downfrom the isoelectric point of insulin, which is around 5.5. However, theinsulin may be less stable at pH significantly above or below 5.5, andfurthermore the pharmaceutically accepted limits of pH 3.0 to 8.5 forinhalation products must be taken into account, since products with a pHoutside these limits may induce irritation and constriction of theairways. To obtain a powder, the solvent must be removed by a processwhich retains the insulin's biological activity. Suitable drying methodsinclude vacuum concentration, open drying, spray drying, and freezedrying. Temperatures over 40° C. for more than a few minutes shouldgenerally be avoided, as some degradation of the insulin may occur.Following the drying step, the solid material can, if necessary, beground to obtain a coarse powder, then, if necessary, micronized.

If desired, the micronized powder can be processed to improve the flowproperties, e.g., by dry granulation to form spherical agglomerates withsuperior handling characteristics, before it is incorporated into theintended inhaler device. In such a case, the device would be configuredto ensure that the agglomerates are substantially deagglomerated priorto exiting the device, so that the particles entering the respiratorytract of the patient are largely within the desired size range.

Where an ordered mixture is desired, the active compound may beprocessed, for example by micronisation, in order to obtain, if desired,particles within a particular size range. The carrier may also beprocessed, for example to obtain a desired size and desirable surfaceproperties, such as a particular surface to weight ratio, or a certainruggedness, and to ensure optimal adhesion forces in the orderedmixture. Such physical requirements of an ordered mixture are wellknown, as are the various means of obtaining an ordered mixture whichfulfills the said requirements, and may be determined easily by theskilled person according to the particular circumstances.

The invention will now be described by way of Examples, which areintended to illustrate but not limit the scope of the invention.

EXAMPLES Comparative Example Therapeutic preparation of insulin, withoutenhancer

Semisynthetic human insulin (Diosynth, 0.8 g) and water (150 ml) wereadded to a beaker. The pH was lowered with 1M HCl to pH 3.4 and thenraised with 1M NaOH to pH 7.4, in order to dissolve the insulin.

Lactose (commercially available, 9.2 g) was added and the pH againadjusted to pH 7.4. The solution was stirred until clear or weaklyopalescent, and concentrated by evaporation, at a temperature of 37° C.over a period of about two days.

The obtained solid cake was crushed, and sieved through a 0.5 mm sieve,and the resultant powder micronised through a jet mill to particles witha diameter of about 2 microns.

EXAMPLE 1 Therapeutic preparation of insulin and sodium caprate; ratio75:25

Semisynthetic human insulin (9.75 g) and water (250 ml) were added to abeaker. The pH was lowered with 1M HCl to pH 3.4 and then raised with 1MNaOH to pH 7.4, in order to dissolve the insulin.

Sodium caprate (Sigma, 3.25 g) was added and the pH again adjusted to pH7.4. The solution was stirred until clear or weakly opalescent, andconcentrated by evaporation, at a temperature of 37° C. over a period ofabout two days.

The obtained solid cake was crushed, and sieved through a 0.5 mm sieve,and the resultant powder micronised through a jet mill to particles ofabout 2 microns diameter.

EXAMPLE 2 Therapeutic preparation of insulin and sodium caprate, withlactose; ratio 50:25:25

Semisynthetic human insulin (7.5 g) was dissolved in water (150 ml) asin Example 1. Sodium caprate (3.75 g) and lactose (3.75 g) were addedand the procedure of Example 1 followed to produce a powder largelyconsisting of particles with a diameter of about 2 microns.

EXAMPLE 3 Therapeutic preparation of insulin and sodium caprate, withlactose; ratio 4:4:92

The procedure of Example 2 was followed, using 0.5 g of semisynthetichuman insulin, 150 ml water, 0.5 g sodium caprate and 11.5 g lactose.

INHALATION STUDIES Study 1

The preparation of Example 1 was used in an inhalation study in twodogs. The preparation was filled into a Wright Dust Feed inhalationapparatus and administered to the dogs. The dosage level was 1 U./kg (1U.=one unit of human insulin=35 μg human insulin, 100%) Blood glucoseand plasma insulin values were measured at various time intervals andthe results are summarised in Tables 1 and 2 below.

                  TABLE I                                                         ______________________________________                                        Blood sample                                                                  time after end                                                                              Blood                                                           of expo       glucose   Insulin conc                                          (minutes)     (mmol/L)  (μU/ml)                                            ______________________________________                                        before        3.9       6.70                                                   0.5          3.6       120.66                                                 5            2.8       194.47                                                 10           2.6       195.39                                                 20           n.d       139.74                                                 22.5         1.6       n.d                                                    31           2.0       73.42                                                  45           1.7       47.49                                                  59.5         1.7       36.21                                                  89.5         2.3       19.28                                                 120           3.0       14.58                                                 240           4.5       5.28                                                  ______________________________________                                         n.d. = not determined                                                    

                  TABLE II                                                        ______________________________________                                        Blood sample                                                                  time after end                                                                              Blood                                                           of expo       glucose   Insulin conc                                          (minutes)     (mmol/L)  (μU/ml)                                            ______________________________________                                        before        3.9       44.84                                                  3            4.2       165.10                                                 6            4.3       158.28                                                 12           3.9       n.d.                                                   14           n.d.      180.72                                                 19           3.0       133.75                                                 30           2.7       143.71                                                 45           2.5       91.62                                                  60           2.4       66.70                                                  90           2.7       38.58                                                 122           3.7       29.15                                                 241           4.1       n.d.                                                  242.5         n.d.      19.76                                                 ______________________________________                                         n.d. = not determined                                                    

The tables illustrate that the insulin/sodium caprate formulationmarkedly increases the plasma level of insulin and decreases the bloodglucose. The peak value for plasma insulin and the minimal value forblood glucose are reached after approximately 15 and 60 minutes,respectively.

Study 2

The preparations of the Comparative Example and Example 1 were eachadministered to four or five dogs, by means of a Wright Dust Feedinhalation apparatus, at a constant dosage level of 1 U./kg. The effectof each formulation on plasma insulin levels and blood glucose levelswas determined at various time points and the results are illustrated inFIGS. 1 and 2. It was found that, while the control formulationcontaining no enhancer produced essentially no change in plasma insulinlevels, the formulation containing both insulin and enhancer produced arise in plasma insulin levels from about 20 μU/ml at time zero to about80 μU/ml 15 min. after inhalation of the powder. Likewise, the controlanimals registered a maximal drop in blood glucose of about 0.5 mmol/lfollowing inhalation of insulin without enhancer, while the animalswhich inhaled insulin plus enhancer registered a transient drop of about1.7 mmol/l, from about 4.0 mmol/l to about 2.3 mmol/l. Thus, insulincombined with the enhancer, sodium caprate, was quickly absorbed intoand cleared from the systemic circulation, with a correspondingtransient decrease in blood glucose levels. In contrast, insulin withcarrier (lactose) but no enhancer was detectably absorbed only to a verysmall degree. (p=0.0002 for insulin/caprate vs. insulin/lactose.)

Study 3

The preparations of Examples 1-3 were each tested at varying dosagelevels in two dogs. Administration of the preparations was by means of aWright Dust Feed inhalation apparatus. Plasma insulin and blood glucoselevels were measured at various time intervals following inhalation. Theresults are indicated in FIGS. 3-6, and show that insulin combined withsodium caprate in various proportions absorbs quickly, and that peakvalues are obtained after 20-30 minutes, followed by correspondingdecreases in blood glucose levels. The results also indicate that byinhalation of insulin powder a plasma profile can be obtained which ismore like the natural physiological profile than the profile obtainedafter subcutaneous injection of insulin.

EXAMPLE 4 Insulin and sodium caprate, 75:25; mixing of micronisedpowders

Biosynthetic human insulin (53 g) was micronised in an Airfilco Jet Mill(Trade Mark, Airfilco Process Plant Limited), with pressurised nitrogen(feed pressure 7 bar, chamber pressure 5 bar), to a mass median diameterof 2.4 micrometers.

Sodium caprate (170 g) was micronised in an Airfilco Jet Mill (TM), withpressurised nitrogen (feed pressure 5 bar, chamber pressure 3 bar), to amass median diameter of 1.6 micrometers.

The micronised biosynthetic human insulin (45 g) and sodium caprate(14.26 g) were dry mixed according to the following procedure: Half ofthe insulin was added to a mixing device comprising a mixing cylinder ofvolume 4.4 liters divided, by a sieve of width 1 mm, into twocompartments, with a metal ring in each compartment to aid mixing andstirring. The sodium caprate and finally the rest of the insulin, wereadded. The mixing cylinder was closed, turned 180 degrees, and mountedin a motorised shaking apparatus. The motor was turned on and shakingcontinued for approximately two minutes, until all the insulin andsodium caprate had passed through the sieve. The motor was turned offand the mixing cylinder turned 180 degrees, again mounted on the shakingapparatus and shaking was again effected until all the powder had passedthrough the sieve. This procedure was repeated a further eight times togive a total mixing time of approximately 20 minutes.

The preparation so obtained was administered to 5 dogs by inhalation,using a Wright Dust Feed inhalation apparatus, at a dosage level of 1U./kg, and the plasma insulin level determined at various time pointsafter administration.

The results obtained were compared with the plasma insulin levelsobtained when biosynthetic insulin, micronised as above to a mass mediandiameter of 2.4 micrometers, were administered to five dogs in the sameway and at the same dosage levels, and with the plasma insulin levelsobtained when a therapeutic preparation of insulin and sodium caprate ina ratio of 90:10 was administered to five dogs in the same way and atthe same dosage levels as above. In this case the therapeuticpreparation was prepared as follows: Human semisynthetic insulin was gelfiltrated to reduce the zinc content from 0.52% to 0.01% relative tocontent of insulin. Insulin (4.5 g) and sodium caprate (0.5 g) weredissolved in water (232 ml). The solution was stirred until clear andthe pH adjusted to 7.0. The solution was concentrated by evaporation at37° C. over a period of about two days. The obtained solid cake wascrushed, and sieved through a 0.5 mm sieve, and the resultant powdermicronised through a jet mill to particles with a mass median diameterof 3.1 micrometers.

The results of these comparisons are presented in FIG. 9. The resultsdemonstrate some improvement in the bioavailability of insulin with the90:10 formulation, and a dramatic improvement in the bioavailability ofinsulin with the 75:25 preparation according to the present invention,as compared to insulin alone. (p=0.0147 for the difference between 75:25and 100:0)

EXAMPLE 5 Selection of enhancers

Each of the compounds listed in Table III was tested for its ability toenhance uptake of insulin, and thus affect blood glucose levels, in arat model. Various forms of insulin were employed: recombinant orsemisynthetic human or bovine. Each formulation was prepared as inExamples 1-3 above, drying and processing the insulin/enhancer solutionto produce an inhalable powder.

The powder was administered to rats by inhalation, and the blood glucoselevels of the rats were subsequently monitored. These levels werecompared to the corresponding values obtained from rats which hadinhaled insulin formulations without enhancer.

                  TABLE III                                                       ______________________________________                                                         Enhancer:Insulin:lac-                                                                        Effe                                          Substance        tose           ct                                            ______________________________________                                        Octylglucopyranoside                                                                             4:4:92       (+)                                           Sodium ursodeoxycholate                                                                          4:4:92       +                                             Sodium taurocholate                                                                              4:4:92       +                                             Sodium glycocholate                                                                              4:4:92       +                                             Lysophosphatidylcholine                                                                          4:4:92       +                                             Dioctanoylphosphatidylch                                                                         2:4:94       (+)                                           oline                                                                         Didecanoylphospatidylcho                                                                         4:4:94       -                                             line                                                                          Sodium             2:4:94       +                                             taurodihydrofusidate                                                          Sodium caprylate   25:75:0      -                                             Sodium caprate     10:90:0      (+)                                           Sodium caprate   17.5:82.5:0    (+)                                           Sodium caprate     25:75:0      +                                             Sodium caprate     4:4:92       +                                             Sodium laurate     25:75:0      (+)                                           Potassium oleate   4:4:92       +                                             Potassium caprate                                                                                27:73:0      +                                             Lysine caprate     35:65:0      +                                             Sodium myristate   30:70:0      +                                             Dimethyl-β-cyclodextrin                                                                     75:25:0      +                                             ______________________________________                                         + effect, i.e. enhancer gives a significant decrease in blood glucose         level                                                                         - no or very small effect                                                     (+)  effect, not as marked as "+                                         

EXAMPLE 6 Selection of enhancers

A standard in vitro assay utilizing an epithelial cell line, CaCo-2(available through the American Type Culture Collection (ATCC),Rockville, Md., U.S.A.), has been developed to assess the ability ofvarious enhancer compounds to promote transport of insulin and othermarkers across an epithelial cell monolayer, as a model for theepithelial cell layer which functions in the lung to separate thealveolus from the pulmonary blood supply. In this assay, the enhancerand insulin or other marker are dissolved in aqueous solution at variousproportions and/or concentrations, and applied to the apical side of thecell monolayer. After 60 min incubation at 37° C. and 95% RH (relativehumidity), the amount of the marker on the basolateral side of the cellsis determined: for example, by use of a radioactively labelled marker.For the particular enhancer (sodium caprate) tested in the experimentsshown in FIGS. 5 and 6, the amount of marker (mannitol, MW 360) whichappears on the basolateral side is dependent upon the concentration ofenhancer used, at least up to 16 mM sodium caprate (FIG. 7). This istrue even when insulin is added to the enhancer/mannitol mixture (1:3sodium caprate:insulin, by weight) (FIG. 8). This concentration ofsodium caprate (16 mM) was also found to promote absorption of insulinacross the cell monolayer. The amount of insulin which passed across themonolayer doubled in the presence of 16 mM sodium caprate, compared tothe amount in the absence of any enhancer. It is expected that at higherconcentrations of sodium caprate, the permeability of the cells will befurther increased; however, the potential cytotoxicity of sodium capratemay prevent the use of substantially higher concentrations of thisparticular enhancer.

This in vitro model of epithelial cell permeability can be used as ascreening tool for rapidly testing any desired enhancer for usefulnessin the methods of the invention.

What is claimed is:
 1. A therapeutic preparation, comprising activecompounds (A) insulin and (B) a substance which enhances the absorptionof insulin in the lower respiratory tract, in the form of a dry powdersuitable for inhalation in which at least 50% of the total mass ofactive compounds consists of (a) particles having a diameter of up to 10microns or (b) agglomerates of such particles.
 2. A therapeuticpreparation as claimed in claim 1, characterised in that the therapeuticpreparation contains only said active compounds.
 3. A therapeuticpreparation as claimed in claim 1, characterised in that the dry powdercontains, in addition to said active compounds, a pharmaceuticallyacceptable carrier.
 4. A therapeutic preparation as claimed in claim 3,characterised in that said carrier consists of particles having adiameter of up to 10 microns such that at least 50% of said dry powderconsists of (a) particles having a diameter of up to 10 microns or (b)agglomerates of such particles.
 5. A therapeutic preparation as claimedin claim 3, characterised in that said carrier consists essentially ofcoarse particles, such that an ordered mixture may be formed betweensaid active compounds and the carrier.
 6. A therapeutic preparation asclaimed in claim 4, in which at least 50% of the dry powder consists of(a) particles having a diameter of between 1 and 6 microns or (b)agglomerates of such particles.
 7. A therapeutic preparation as claimedin claim 1 or claim 5, in which at least 50% of the total mass of activecompounds (A) and (B) consists of particles having a diameter of between1 and 6 microns.
 8. A therapeutic preparation as claimed in claim 1,characterised in that the insulin is bovine, porcine, biosynthetic orsemisynthetic human insulin, or a biologically active derivative ofhuman insulin.
 9. A therapeutic preparation as claimed in claim 8,characterised in that the insulin is semisynthetic human insulin.
 10. Atherapeutic preparation as claimed in claim 8, characterised in that theinsulin is a biosynthetic human insulin.
 11. A therapeutic preparationof insulin as claimed in claim 1, characterised in that the substancewhich enhances the absorption of insulin in the lower respiratory tractis a substance which promotes the absorption of insulin through thelayer of epithelial cells in the lower respiratory tract and into theadjacent pulmonary vasculature.
 12. A therapeutic preparation as claimedin claim 11, characterised in that the substance which enhances theabsorption of insulin in the lower respiratory tract is a surfactant.13. A therapeutic preparation as claimed in claim 11, characterised inthat the substance which enhances the absorption of insulin in the lowerrespiratory tract is an anionic surfactant.
 14. A therapeuticpreparation as claimed in claim 11, characterised in that the substancewhich enhances the absorption of insulin in the lower respiratory tractis a bile salt or a bile salt derivative.
 15. A therapeutic preparationas claimed in claim 11, characterised in that the substance whichenhances the absorption of insulin in the lower respiratory tract is aphospholipid.
 16. A therapeutic preparation as claimed in claim 11,characterised in that the substance which enhances the absorption ofinsulin in the lower respiratory tract is an alkyl glycoside.
 17. Atherapeutic preparation as claimed in claim 11, characterised in thatthe substance which enhances the absorption of insulin in the lowerrespiratory tract is a cyclodextrin or derivative thereof.
 18. Atherapeutic preparation as claimed in claim 11, characterised in thatthe substance which enhances the absorption of insulin in the lowerrespiratory tract is the salt of a fatty acid.
 19. A therapeuticpreparation as claimed in claim 11, characterised in that the substancewhich enhances the absorption of insulin in the lower respiratory tractis a salt of capric acid.
 20. A therapeutic preparation as claimed inclaim 11, characterised in that the substance which enhances theabsorption of insulin in the lower respiratory tract is sodium caprate.21. A therapeutic preparation comprising active compounds (A) insulinand (B) sodium caprate, which preparation is in the form of a dry powdersuitable for inhalation, in which at least 50% of the total mass ofactive compounds (A) and (B) consists of (a) primary particles having adiameter of less than 10 microns, or (b) agglomerates of such particles.22. A therapeutic preparation as claimed in claim 21, consistingessentially of said active compounds.
 23. A therapeutic preparation asclaimed in claim 21, characterised in that the preparation comprises, inaddition to said active compounds, a pharmaceutically acceptablecarrier.
 24. A therapeutic preparation comprising insulin, sodiumcaprate and a pharmaceutically acceptable carrier, which preparation isin the form of a dry powder suitable for inhalation, of which at least50% by mass consists of (a) particles having a diameter of less thanabout 10 microns, or (b) agglomerates of said particles.
 25. Atherapeutic preparation, comprisingactive compounds (A) insulin and (B)sodium caprate wherein at least 50% of the total mass of activecompounds (A) and (B) consists of particles having a diameter of lessthan about 10 microns, and a pharmaceutically acceptable carrier,whichpreparation is in the form of a dry powder suitable for inhalation inwhich an ordered mixture is formed between the active compounds and thepharmaceutically acceptable carrier.
 26. A therapeutic preparation asclaimed in claim 1 or claim 21, characterised in that the ratio of (A)to (B) in said preparation is in the range 9:1 to 1:1.
 27. A therapeuticpreparation as claimed in claim 26, characterised in that said ratio isin the range 5:1 to 2:1.
 28. A therapeutic preparation as claimed inclaim 26, characterised in that said ratio is in the range 4:1 to 3:1.29. A therapeutic preparation as claimed in claim 3 or claim 23,characterised in that said carrier is selected from mono-, di-, andpolysaccharides, sugar alcohols and other polyols.
 30. A therapeuticpreparation as claimed in claim 3 or claim 23, characterised in thatsaid carrier is a non-reducing sugar.
 31. A therapeutic preparation asclaimed in claim 30, characterised in that said carrier is raffinose,melezitose, lactitol, maltitol, trehalose, sucrose, mannitol or starch.32. A dry powder inhaler device containing the therapeutic preparationof claim 1 or claim
 21. 33. A single dose, breath actuated, dry powderinhaler for single usage, containing a therapeutic preparation forinhalation, which preparation comprises active compounds (A) insulin and(B) sodium caprate and is in the form of a dry powder in which at least50% of the total mass of active compounds (A) and (B) consists ofparticles having a diameter of up to 10 microns.